Seize the engine: Emerging cell cycle targets in breast cancer

Abstract Breast cancer arises from a series of molecular alterations that disrupt cell cycle checkpoints, leading to aberrant cell proliferation and genomic instability. Targeted pharmacological inhibition of cell cycle regulators has long been considered a promising anti‐cancer strategy. Initial attempts to drug critical cell cycle drivers were hampered by poor selectivity, modest efficacy and haematological toxicity. Advances in our understanding of the molecular basis of cell cycle disruption and the mechanisms of resistance to CDK4/6 inhibitors have reignited interest in blocking specific components of the cell cycle machinery, such as CDK2, CDK4, CDK7, PLK4, WEE1, PKMYT1, AURKA and TTK. These targets play critical roles in regulating quiescence, DNA replication and chromosome segregation. Extensive preclinical data support their potential to overcome CDK4/6 inhibitor resistance, induce synthetic lethality or sensitise tumours to immune checkpoint inhibitors. This review provides a biological and drug development perspective on emerging cell cycle targets and novel inhibitors, many of which exhibit favourable safety profiles and promising activity in clinical trials.

F I G U R E 1 Graphical simplification of cell cycle phases, checkpoints and regulatory mechanisms in breast cancer.mitotic checkpoints.TNBC tumours also display high levels of genomic instability and aneuploidy secondary to the abrogation of DNA repair mechanisms and deficient function of the SAC.The knowledge of these molecular features, along with the clinical benefit observed with CDK4/6 inhibitors (CDK4/6i) in HR + /HER2 − -BC, provide a rationale for targeting cell cycle regulators in patients with relapsed or metastatic disease that are generally considered incurable and for which additional effective therapies are needed.These approaches are based on diverse mechanistic insights including forcing cancer cells to permanently exit the cell cycle or, conversely, to override checkpoints, impairing replication stress tolerance or inducing catastrophic genomic instability.Here, we review the key drug development efforts to target the cell cycle machinery with a focus on the lessons learned and potential applications in BC.

CDK4/6
CDK-cyclin complexes are universal drivers of cell cycle transitions.CDK4/6i are the only approved drugs directly targeting the cell cycle in BC, where they force cancer cells to exit the cell cycle into quiescence.Their development has been focused on HR + BC, where underlying genomic (e.g.4][15][16][17][18][19][20][21][22][23][24] These drugs are United States Food and Drug Administration (US FDA) and European Medicines Agency approved for the treatment of HR + /HER2 − -mBC in combination with endocrine therapy (ET), while abemaciclib is also approved as monotherapy after progression to ET and chemotherapy.Dalpiciclib, which has been studied in China, also led to survival improvements in combination with fulvestrant in endocrine-resistant disease. 25,26n the early BC setting, abemaciclib was recently approved in combination with ET for the adjuvant treatment of high risk early HR + /HER2 − BC following the results of the MonarchE trial, and initial reports of the adjuvant NATALEE ribociclib trial are positive. 27,28In contrast, the adjuvant palbociclib trial failed to meet its primary endpoint of improving invasive disease-free survival (IDFS).
A variety of trials have explored the impact of adding CDK4/6i to neoadjuvant ET on the pathological complete response rate as well as on other surrogate endpoints (e.g.0][31][32][33][34][35] While these neoadjuvant trial results are encouraging, the lack of event-free survival data and the heterogeneity in trial design, particularly regarding the use of different early biological endpoints, preclude the comparison among agents and translation to clinical practice. In sum, while all CDK4/6i prolong progression-free survival (PFS), palbociclib has not shown benefits in overall survival (OS) and IDFS.Whether this reflects true differences between these agents, or rather in the trial designs, is unclear. 36,37In this regard, palbociclib exhibits comparable potency against cyclin D1/CDK4 and cyclin D2/CDK6, whereas ribociclib and abemaciclib display greater potency against CDK4, with abemaciclib also blocking CDK1, CDK2, CDK5 and CDK9.Additionally, the continuous dosing of abemaciclib differs from the 21 days on, 7 days off regimen of its counterparts, potentially influencing the development of biologically distinct resistant populations.][40] Other CDK4/6i, such as lerociclib or trilaciclib, remain largely investigational at present.Lerociclib, a continuous oral CDK4/6i, has shown a safety profile and preliminary efficacy comparable to the approved agents in HR + /HER2 − -mBC. 41The development of trilaciclib, which aims to maintain immune and bone marrow cells in G1 arrest and protect them from chemotherapy-induced damage, has mostly focused on TNBC, where it has been granted an US FDA fast track designation, and is currently being evaluated in the phase 3 PRESERVE 2 study. 42,43ntrinsic resistance to CDK4/6i is uncommon but acquired resistance to therapy eventually emerges in most patients.Identifying effective therapies following progression on CDK4/6i in HR + /HER2 − -BC patients is an area of urgent clinical need considering the limited activity of single-agent fulvestrant following progression. 44,45While acquired CDK4/6i cross-resistance is frequent in BC models, there is preclinical evidence supporting the use of abemaciclib after progression on palbociclib. 46A retrospective, multi-centre cohort of 87 patients treated with abemaciclib after progression to palbociclib reported a 6-month PFS of 37%, and a randomised phase 3 trial of this strategy is ongoing. 47Consistently, the MAINTAIN phase II trial reported that the combination of ET plus ribociclib showed a modest benefit over fulvestrant monotherapy after progression on first-line ET plus CDK4/6i (84% of patients had received palbociclib), adding to the potential role for CDK4/6i switching following progression. 48In contrast, the PACE study, evaluating palbociclib added to fulvestrant after progression on an AI plus CDK4/6i (mostly palbociclib), failed to show benefit. 49The question of whether the utility of continuation of a CDK4/6i-based strategy following initial progression depends on the biology of resistant disease is just beginning to be explored.

Mechanisms of resistance to CDK4/6i
As more patients have been treated with CDK4/6i, correlative analyses have shed light on potential resistance mechanisms.From a mechanistic standpoint, genomic alterations associated with resistance may be divided into two categories: resistance through the disruption of the cell cycle machinery (including alterations in RB1, CCNE1, CDK6, CDK4, p16, CDK2, CDK7, CCND1 or INK) and resistance through compensatory upstream signalling (alterations in PI3K, PTEN, FGFR1/2, FAT1/HIPPO, ERBB2 or RAS signalling) (Figure 1). 50ost of the efforts regarding targeted therapy to date have focused on inhibiting the PI3K/AKT/mTOR pathway, based on work that was underway before the widespread introduction of CDK4/6i use in the first line.As activating mutations in PI3K tend to be truncal rather than acquired alterations, and thus are not specifically acquired with CDK4/6i resistance, the activity of these agents following progression on CDK4/6i could be influenced by ongoing dependence on this oncogenic signalling in resistant disease.With the initial evaluation of the PI3Kα-selective inhibitor alpelisib conducted in patients mostly untreated with CDK4/6i, the phase 2 BYLieve trial was carried out to evaluate alpelisib in CDK4/6i pre-treated participants.Notably, this trial reported a 6-month PFS of nearly 50%. 51he confirmatory phase 3 randomised EPIKB5 trial was recently launched.A phase 1/2 trial evaluating inavolisib, an investigational PI3Kα-selective inhibitor, in combination with fulvestrant, recently showed a favourable safety profile compared to other agents of the same class and an encouraging preliminary anti-tumour activity with an overall response rate (ORR) of 25% and a clinical benefit rate (CBR, defined as the sum of complete response, partial response and stable disease > 6 months) of 49% in a similar population of patients with PIK3CA-mutated HR + /HER2 − -BC following progression on ET and CDK4/6i. 52Alpelisib and inavolisib will be compared in this setting, in combination with fulvestrant, in the randomised phase 3 INAVO121 study. 53Data from ongoing studies testing mutant-selective PI3K inhibitors LOXO783 or RLY-2608 that may provide a more favourable therapeutic index are also eagerly awaited.
Within the same pathway, the phase 3 CAPItello-291 trial recently reported that the addition of the AKT inhibitor capivasertib to fulvestrant improved PFS in endocrineresistant patients, including 60% who had progressed on a CDK4/6i. 54The ongoing phase 3 FINER study testing ipatasertib will provide further data about the benefit of an AKT inhibitor in the immediate post-CDK4/6i setting.Additionally, there is encouraging retrospective data supporting the similar benefit in survival outcomes of exemestane plus mTOR inhibitor everolimus irrespective of prior CDK4/6i exposure, and the benefit of the triplet exemestane, everolimus and ribociclib beyond CDK4/6i progression is currently being investigated in the phase 1/2 TRINITI-1 trial. 55,56he pan-HER2 inhibitor neratinib and FGFR1-4 kinase inhibitor rogaratinib for patients with ERBB2 mutations or FGFR1/2/3 amplification, respectively, are other examples of targeting compensatory upstream signalling in the post-CDK4/6i setting, and combinations with other agents, including CDK4/6i, are being developed. 57,58

CDKs beyond CDK4/6
The components of the CDK family can be functionally classified into those directly intervening in the cell cycle process (CDK1, CDK4, CDK5, CDK6) and those acting as transcriptional regulators (CDK7, CDK8, CDK9, CDK11, CDK19, CDK20; of which only CDK7, CDK11 and CDK20 act as regulators of cell cycle progression). 59A number of pan-CDK inhibitors (CDKi) have been studied over the last two decades with generally limited anti-tumour activity and considerable toxicity (Figure 2 and Table 1).The first non-selective CDKi to enter clinical testing, alvocidib, was shown to produce potent cell cycle arrest and apoptosis in preclinical models. 60However, alvocidib showed minimal clinical anti-tumour efficacy as a single agent in solid tumours, while combination trials with docetaxel or cisplatin produced intolerable neutropenia or gastrointestinal side effects. 61,62][69] The recent success of CDK4/6i and the improved understanding of genomic vulnerabilities spurred a new wave of development of selective CDKi in solid tumours.1][72] While the cyclin E-CDK2 complex participates in the G1/S phase transition through RB1 phosphorylation, CDK7 exerts a wider role in cell cycle progress by acting as a CDK-activating kinase (Figure 1). 735][76][77] Clinical trials with compounds selectively blocking CDK2 or CDK4 are ongoing, some of them as combinations with ET and CDK4/6i in HR + /HER2 − -mBC.In the dose escalation part of the phase 1/2a trial testing the first-in-class CDK2/4/6i PF-06873600 (ebvaciclib), an acceptable safety profile was observed along with preliminary evidence of anti-tumour activity in 59 HR + /HER2 − -BC patients who had progressed on a previous CDK4/6i and ≤2 prior chemotherapy lines. 78This included the achievement of a disease control rate (DCR, indicating the achievement of complete response, partial response or stable disease as best response) of 48% (28 out of 58) in the monotherapy arm and 67% (six out of nine) in combination with fulvestrant. 78However, ebvaciclib is no longer advancing in BC, and the development of another CDK2/4/6i, NUV-422, was terminated early due to the unexpected observation of uveitis during dose escalation. 79ore selective agents targeting CDK2 or CDK4 have recently produced promising results (Table 1).For instance, the selected CDK2i PF-07104091 elicited a 19% ORR and 62% DCR among 16 heavily pre-treated post-CDK4/6i HR + /HER2 − -mBC patients; however, there were notable toxicities including 77% all-grade nausea (14% ≥G3), 50% diarrhoea (9% ≥G3) and 46% fatigue (20% ≥G3). 80In a similar setting, CDK2i BLU-222 is undergoing dose escalation and has shown a partial response in one out of 12 BC patients, a case of HR + /HER2 − -mBC that had received palbociclib, abemaciclib and capecitabine. 81ore favourably, among 21 evaluable patients treated with the selective CDK4i PF-07220060, ORR was 29% and CBR was 54%, with better tolerability including 50% all grade diarrhoea (0% ≥G3), 50% neutropenia (15% ≥G3) and 39% nausea (4% ≥G3). 82he first-in-class selective CDK7 inhibitor, intravenous SY-1365, exhibited frequent gastrointestinal toxicity and limited activity and its development was terminated to prioritise oral SY-5609. 83-85SY-5609 has been safe and tolerable in a variety of dosing schedules, and its combination with fulvestrant has so far resulted in a CBR of 25%, with no responses, among 12 evaluable patients with heavily pre-treated, post-CDK4/6i HR + /HER2 − -mBC. 85he molecule most advanced in clinical testing is CT7001 (samuraciclib), which recently reported safety and preliminary efficacy data from a modular phase 1/2a trial including post-CDK4/6i HR + /HER2 − -mBC and TNBC patients.Samuraciclib's adverse event profile included frequent, low-grade gastrointestinal toxicities such as diarrhoea (90%), nausea (81%) and vomiting (52%).In  1 and 2, including drugs with no available clinical data at present.

TA B L E 1
Drug development targeting cyclin-dependent kinases (CDKs) beyond CDK4/6.Clinical evidence and ongoing trials are presented, with a special focus on those with breast cancer cohorts.combination with fulvestrant, a 24-week CBR of 39%, including two partial responses (8%), was reported among 24 evaluable HR + /HER2 − -mBC patients. 86In the TNBC cohort, composed of 21 evaluable women who had received prior taxane and anthracycline, samuraciclib achieved a 24-week CBR of 24%, including one (5%) partial response. 87 combination of samuraciclib with the oral selective ER degrader (SERD) giredestrant is currently being tested within the MORPHEUS phase 1/2b umbrella study, and combinations with elacestrant and ER proteolysis targeting chimera ARV-471 are also planned.Other CDK7i recently in development include LY3405105 and XL-102, with both showing a lower incidence of all-grade gastrointestinal toxicity and mild myelotoxicity, but limited anti-tumour activity including ORR 0% with the former agent.88,89 While the improved toxicity profile is encouraging, more data on anti-tumour activity are awaited.
1][92][93] In particular, more than 15% of breast tumours harbour alterations in CDK8/19, mostly copy number gains, and its expression is inversely correlated with ER expression and relapse-free survival. 91

S PHASE
Entry into S phase is essentially controlled by the CDK2/cyclinA complex, where CDK2 is activated by CDK4/6-dependent E2F upregulation and by CDK7, and inhibited by DNA damage-mediated cdc25A and WEE1 (Figure 1).The DNA-damage response pathway is thus critical for cell cycle regulation in the S but also G2-M checkpoints.In particular, DNA double-strand breaks activate the ATM-CHK2-cdc25A axis that prevents S phase entry, while single-strand DNA damage induces the ATR-CHK1-cdc25A axis that limits G2/M progression, with substantial overlapping of the two pathways including WEE1 activation that blocks CDK2 (S phase checkpoint) and CDK1 (G2/M checkpoint).These proximal DNA damage-induced regulatory pathways and their various effects on the cell cycle have been reviewed elsewhere and fall beyond the scope of our review, where we aimed to focus on more distal kinases amenable to drug development. 94,95 key milestone of the S phase is centrosome duplication.Centrosomes are the microtubule organising centres in eukaryotic cells and are composed of two barrel-shaped organelles embedded in a matrix of proteins known as pericentriolar material.Their duplication in the S phase is critical for the equal distribution of genetic material in mitosis.This process is governed by highly conserved molecular mechanisms that prevent the generation of multipolar spindles and subsequent aneuploidy and genomic instability.In humans, centriole initiation requires PLK4 serine/threonine kinase activity (Figure 1). 96Although the specific underlying mechanisms remain incompletely understood, PLK4 provides a mitotic checkpoint to sense DNA damage and other cellular abnormalities.
The identification of PLK4 as a promising therapeutic target resulted from a systematic approach combining kinome-wide RNAi screening and gene expression analysis in cell lines and human BC. 97][99][100][101] This is likely explained by centrosome amplification, multipolarity and resulting aneuploidy and genomic instability.PLK4 upregulation is associated with a higher incidence of lymph node metastasis, distant metastasis, shorter survival and worse response to neoadjuvant taxane-based chemotherapy and adjuvant tamoxifen. 98,102,103,104everal small-molecule PLK4 inhibitors have been developed with varying selectivity (Figure 2 and Table 2).These include CFI-400945, centrinone, centrinone B and YLT-11, all of which interact with the ATP-binding pocket in the catalytic domain of PLK4.Of these, CFI-400945, has proceeded to clinical development as a first-in-class oral PLK4 inhibitor, identified through an academic discovery program.Anti-tumour activity of CFI-400945 was demonstrated in human BC xenografts representing all recognised BC subtypes. 97CFI-400945 blocks autophosphorylation of PLK4, which is a critical step for its activation, and its anti-tumour effects may depend on facilitating errors in chromosome segregation and genomic instability.Recently, our group demonstrated the synergistic anti-proliferative effect of CFI-400945 in combination with radiotherapy in TNBC models. 105Centrinone A and B are the most selective PLK4 inhibitors and have been extensively studied as preclinical tool compounds, but lack pharmacologic properties for clinical drug development. 106,107ast, YLT-11, the most recently described small-molecule inhibitor, has shown high PLK4 selectivity and remarkable anti-proliferative activity in vitro and in human BC xenografts. 108o date, only CFI-4000945 has been tested in clinical trials.Its first-in-human trial enrolled 52 patients with advanced solid tumours including four BC patients. 109An acceptable safety profile was reported, including fatigue (37%), nausea (21%) and neutropenia (21%), generally of low-grade.Dose-dependent neutropenia was observed, of grade ≥3 in 19% at the highest dose level. 109Although there was limited anti-tumour activity in the phase 1 trial TA B L E 2 Drug development targeting non-cyclin-dependent cell cycle kinases.Clinical evidence and ongoing trials are presented, with a special focus on those with breast cancer cohorts.that included refractory solid tumours (ORR 2%, CBR 8%), the favourable tolerability profile and strong preclinical rationale led to further therapeutic development in solid tumours across four phase 2 trials, including two BC trials.Preliminary data from a phase 2 study investigating CFI-400945 monotherapy were recently presented.Among 27 HR + /HER2 − -mBC patients with a median of three prior therapies, including 75% with prior CDK4/6i, three (11%) achieved partial response and seven (26%) continued on treatment beyond 6 months, with a 33% incidence of grade ≥3 neutropenia but otherwise good clinical tolerability. 110mong 13 patients in the TNBC cohort, one patient had stable disease for more than 6 months, but no objective responses were observed.These findings are consistent with another phase 2 study that tested CFI-400945 in combination with the PD-L1 immune checkpoint inhibitor durvalumab in unselected, pre-treated TNBC patients.In this trial, two (15%) out of 13 patients had a short-lived stable disease as best response. 111Overall, CFI-400945 continues its clinical development in HR + /HER2 − -mBC and the results from its correlative program are awaited to identify those patients who can derive the most benefit.

G2/M PHASE TRANSITION
The initiation of mitosis is ultimately regulated by the activation of the CDK1/cyclin B complex.DNA damage and replication stress checkpoints prevent the accumulation and propagation of genetic errors during cell division.DDR-related kinases such as ATM and CHK2, and ATR and CHK1, prevent the G2/M transition by activating serine-threonine kinases WEE1 and PKMYT1, which maintain inhibitory phosphorylation of CDK1 (Figure 1). 112onversely, the degradation of both WEE1 and PKMYT1 is mediated by their phosphorylation by CDK1 and PLK1 in the absence of DNA damage. 113,114CDK1/cyclin B complex activation is also critically regulated by aurora kinase A (AURKA), which additionally activates PLK1 to promote mitosis entry.

WEE1
WEE1 and PKMYT1 have been proven essential for cancer cell viability in CRISPR-Cas9 screens and are consistently found upregulated in solid tumours, where they are thought to maintain tolerable levels of genetic instability. 115,116There is, however, scarce data about their role as prognostic biomarkers, with their expression linked to more aggressive phenotypes and worse outcomes in gene datasets of TN and HR + BC. [117][118][119] The potential of WEE1 as a therapeutic target in BC was substantiated more than a decade ago through RNAi screens of the human kinome. 120,121Several WEE1 inhibitors have been developed with the goal of causing mitotic catastrophe by abrogating the G2/M checkpoint, thereby forcing entry into mitosis despite an intolerable burden of genomic damage.
The first compounds, PD0166285 and PD0407824, exhibited modest anti-proliferative effect and reduced selectivity, and did not reach clinical development. 122,123Most of the available preclinical and clinical studies on WEE1 targeting in BC have used adavosertib, a highly selective, ATP-competitive, small-molecule inhibitor.5][126] Moreover, the higher expression of cyclin E (CCNE) has been described as a predictive biomarker for adavosertib monotherapy in TNBC cell lines. 124These studies emphasise the potential use of WEE1i to generate homologous recombination deficiency (HRD) and (re)sensitise tumours to agents targeting DNA or its repair machinery.More recent data suggest that WEE1 may also play a prominent role in CDK4/6i-resistant HR + /HER2 − -mBC.By means of siRNA or adavosertib use, WEE1 abrogation significantly decreased cell proliferation and induced apoptosis and G2/M arrest in several endocrineand CDK4/6i-resistant cell lines, an effect observed independent of the RB1 status. 71,118,127our WEE1i, adavosertib, Debio 0123, IMP7068 and ZN-c3, have entered clinical trials (Figure 2 and Table 2).Adavosertib was first explored in two different phase 1 trials in monotherapy and in combination with gemcitabine, cisplatin or carboplatin. 128,129In one of them, two out of eight patients carrying BRCA1/2 mutations, none of them with BC, showed confirmed partial responses.From a total of 25 patients enrolled, common toxicities included myelosuppression (40%) and nausea, vomiting and diarrhoea (60%), mostly of grade ≤2. 128In another phase 1 trial, adavosertib was given with standard chemotherapy including gemcitabine or platins. 129Of 176 patients evaluable for efficacy, 94 (53%) had stable disease as best response and 17 (10%) achieved a partial response.Further in development, Keenan et al. explored the combination of adavosertib and cisplatin as first-or second-line therapy for mTNBC in a phase 2 trial, where ORR was 26%, CBR was 32% and median PFS was 4.9 months, largely similar to reports of platinum monotherapy in this population in the TNT and TBCRC009 trials. 130,131The most common treatment-related adverse events (TEAEs) were nausea (50%), diarrhoea (35%) and neutropenia (29%), including grade ≥3 diarrhoea (21%) and neutropenia (18%). 132This study included an effort to capture correlative biomarkers of response and found that increased post-treatment CD3/CD4 + and CD3/CD8 + T-cell numbers were associated with improved clinical outcome, while no previously reported WEE1i transcriptomic correlates were identified. 132,133The strategy of combining WEE1 and PARP inhibition was explored in the phase 2 VIOLETTE trial, which coupled adavosertib and olaparib in mTNBC. 134However, this combination was terminated early due to substantial myelotoxicity, and adavosertib development was subsequently discontinued due to its narrow therapeutic window.
While the development of adavosertib has focused principally on ovarian cancer, where it has reached phase 2 testing in combination with chemotherapy, there are other WEE1i with similar preclinical activity and expected superior safety profiles based on ongoing phase 1 trials. 1357][138][139] Similarly, Debio 0123 has been shown tolerable with three grade ≥3 thrombocytopenia events among 38 patients as the most frequent high-grade TEAE. 140These novel inhibitors will eventually inform the potential of WEE1 as a pharmacological target in BC.

PKMYT1
Less studied than WEE1 itself, the related family member PKMYT1 recently emerged as a therapeutic target through a genome-scale CRISPR-Cas9-based synthetic lethality screen in cellular models of CCNE1 amplification. 141Following this observation, a selective, orally bioavailable inhibitor was identified, RP-6306, which has been shown to potently inhibit CDK1/cyclin B activity and produce DNA damage.These findings occurred in a dose-and timedependent fashion in tumour xenograft models including the implantation of the CCNE1-amplified TNBC cell line HCC1569. 141Moreover, the addition of gemcitabine rendered a synergistic effect in the context of DNA replication stress and extended S phase caused by CCNE1 overexpression.
In an analysis of the kinomes of a series of 20 HR + BC PDX tumours, Chen et al. 119 recently described PKMYT1 as a marker of hormone independent growth and poor outcome.Of note, PKMYT1 was found significantly decreased after oestradiol deprivation, an effect likely governed by the oestrogen response elements contained in the regulatory region of the PKMYT1 gene.These regulatory elements are absent in the WEE1 gene, thereby suggesting a potential biological rationale for PKMYT1 inhibition over WEE1 in HR + tumours.Altogether, these findings hold promise for further development in BC, particularly where synthetic lethality can be exploited and in luminal BC.RP-6306 recently entered first-in-human clinical studies as monotherapy and in combination with gemcitabine, FOLFIRI and an ATRi, and the gemcitabine combination is also being evaluated in endocrine-resistant HR + /HER2 − -mBC, with integrated evaluation of putative synthetic lethal biomarkers (Table 2).

Aurora kinases
Once the threshold levels of CDK1 activity are reached, entry into mitosis is triggered by the phosphorylation of a large number of CDK1 substrates, including PLK1, Aurora A (AURKA) and Aurora B (AURKB) mitotic kinases. 142urora kinases are a family of serine/threonine kinases that play critical functions during mitosis and are consistently overexpressed in malignant tumours.The most studied component is AURKA, which controls the G2/M transition but also exerts pleiotropic functions in centrosome maturation, cytokinesis and the modulation of key transducers of oncogenic signalling. 143In contrast, AURKB is essentially involved in chromosome alignment, kinetochore-microtubule attachment and cytokinesis.AURKA expression, which is strongly correlated with other markers of proliferation, has been shown to predict worse survival outcomes in both HR + /HER2 − and TNBC subtypes. 144,145AURKA upregulation has been described in endocrine-resistant HR + models, where its inhibition restored sensitivity to ER blockade and increased the efficacy of ET. [146][147][148][149] At the genomic level, AURKA amplification has been recently reported as a mechanism of resistance to CDK4/6i, and, interestingly, AURKA inhibition has demonstrated a synthetic lethal interaction with RB1 loss, a well-known mechanism of acquired resistance to CDK4/6i. 150,1513][154] Furthermore, our group recently showed the enhanced sensitivity to AURKAi alisertib, barasertib and tozasertib, of a panel of palbociclib-resistant BC cells, characterised by an increased incidence of micronuclei and segregation errors. 155any drugs targeting aurora kinases have entered clinical development for solid tumours, most of which have been discontinued in early stages due to modest activity, toxicity or to focus in haematological malignancies (Figure 2 and Table 2). 156Dose escalation of pan-aurora kinase inhibitors were limited by frequent toxicities, including neutropenia, fatigue, diarrhoea and hypertension.While selective inhibitors displayed a more favourable safety profile, the lack of anti-tumour responses as single agents diminished enthusiasm for further development. 157lisertib is the only AURKAi currently in development in BC.Early phase studies evaluating alisertib alone or in combination with fulvestrant in endocrine-resistant HR + /HER2 --mBC demonstrated a favourable safety profile and promising anti-tumour activity. 158Three phase 2 trials have tested alisertib alone or in combination with fulvestrant or paclitaxel in advanced BC. 159,160 In the first phase 2 study, alisertib monotherapy demonstrated a manageable safety profile in a cohort of 53 BC patients, with an ORR of 18% in HR + or HER2 + BC patients resistant to ET, but minimal activity in TNBC. 161In the phase 2 study by Haddad et al., 162 91 patients who had previously received ET and CDK4/6i were treated with alisertib alone or in combination with fulvestrant.For alisertib monotherapy, ORR was 20%, 24-week CBR was 41%, and median PFS was 5.6 months; all similar to the combination where ORR was 20%, CBR was 29% and median PFS was 5.4 months. 162The correlative analyses published with this study are based solely on ER and AURKA expression in pre-treatment biopsies, where a positive AURKA expression was significantly associated with a shorter PFS in the monotherapy arm but not in the combination arm, and with additional studies underway. 162Alisertib in combination with paclitaxel has been compared with paclitaxel monotherapy in 139 HR + /HER2 − endocrine-resistant patients, only 20% of whom had received prior CDK4/6i.ORR was similar in both treatment arms (31 vs. 34%), while median PFS was significantly longer with the combination (10.2 vs. 7.1 months). 160Altogether these trials demonstrate that alisertib is tolerable but associated with an incidence of grade ≥3 neutropenia ranging from 40% in monotherapy to 60% in combination with paclitaxel.Despite the potential interest of further development in the post-CDK4/6i treatment landscape, no trials in this population are currently registered.

TTK
The segregation of sister chromatids during mitosis is under the mechanical control of the mitotic spindle, a highly conserved apparatus nucleated by the centrosomes and composed of microtubules and motor proteins.Once that chromosomal kinetochores are attached to the spindle microtubules and aligned at the metaphase plate, the E3 ubiquitin ligase activity of the anaphase-promoting complex (APC) governs the progression of anaphase and completion of mitosis. 177In the presence of chromatid missegregation, the APC is inhibited by the SAC (Figure 1). 178ne of the critical regulatory steps is the recruitment of TTK, also known as Monopolar spindle 1 (MPS1), a serine/threonine and tyrosine kinase, to unattached kinetochores, where it activates the SAC to block mitosis. 179hese observations highlight TTK as an appealing target for drug development, where its inhibition can induce premature anaphase, aneuploidy and intolerable genomic instability.][182] However, its role as a biomarker remains unclear.
3][184][185][186] Given the mechanistic rationale of combining TTKi, which override the SAC, with taxane-based chemotherapy, which engages this checkpoint, substantial preclinical and clinical investigation has focused on such combinations.Several ATP-competitive TTKi have been studied in preclinical models and a few have reached early phase clinical trials.][189] Clinical data is available from BAY1161909, BAY1217389, CFI-402257 and S81694, with a particular focus on their combination with paclitaxel (Figure 2 and Table 2).The first clinical evidence of TTK inhibition in patient cohorts was provided by the phase 1 trial evaluating the lead compound BAY1161909 in combination with paclitaxel. 190espite an acceptable safety profile and signs of antitumour activity, with confirmed responses in 14% of patients and DCR ranging from 43 to 60% depending on the dose of paclitaxel (75 vs. 90 mg/m 2 ), BAY1161909 was deprioritised in favour of BAY1217389. 190Phase 1 data from the combination of the follow-up compound and paclitaxel in 75 patients, one third of whom had BC, showed concerning bone marrow toxicity, with grade ≥3 neutropenia in 32% of patients and febrile neutropenia in 16% of patients. 191espite an ORR of 32% and DCR of 78%, the myelotoxicity of the combination may limit further development.
Another orally active TTKi, CFI-402257, has shown preclinical monotherapy and taxane-combination activity in HR + /HER2 − and TNBC models. 184,192,193Of note, enhanced cytotoxicity has been observed in a subset of CDK4/6i-resistant models, including those harbouring loss of RB1. 155,184,193Currently, two phase 1/2 trials are underway evaluating CFI-402257 in advanced solid tumours and BC.One of them is evaluating CFI-402257 in monotherapy and in combination with fulvestrant, integrating a dose escalation part in patients with advanced solid tumours and three expansion cohorts at the recommended phase 2 dose (RP2D) including TNBC and HR + /HER2 − -mBC patients progressing on AI and CDK4/6i. 194A favourable toxicity profile was reported from 66 patients enrolled across dose escalation and expansion cohorts, 25 of whom had BC, including lowgrade fatigue (47%), nausea (46%) and diarrhoea (32%).Only 9% of patients experienced all-grade neutropenia, 6% being grade ≥3, the latter occurring in patients allocated to the declared RP2D dose or higher. 194The overall 6-month CBR was 12% and ORR was 5%; however, all the 3 patients with confirmed responses had HR + /HER2 − -mBC, with an average of 7 prior systemic therapies including CDK4/6i.In combination with fulvestrant in 20 HR + /HER2 − patients, ORR was 10% and 6-month CBR 25%, with prolonged benefit observed in several patients.In a separate trial testing the combination of CFI-402257 and paclitaxel, safety and preliminary efficacy results were recently presented from 29 HER2 − BC patients, including 90% HR + of whom 75% had previously been treated with CDK4/6i. 195,196The ORR was 8%, CBR was 55% and, consistent with the known toxicity profile of paclitaxel, the most frequent clinical adverse events included fatigue (72%), nausea (52%), diarrhoea (45%) and peripheral neuropathy (45%).As expected with weekly paclitaxel, neutropenia was almost universal; grade ≥3 neutropenia was observed in 70% of patients with some dose-dependency.Following its manageable safety profile and encouraging preliminary efficacy in heavily pre-treated HR + /HER2 − -mBC, an ongoing study is further evaluating CFI-402257 in combination with fulvestrant in HR + /HER2 − -mBC after disease progression to ET plus CDK4/6i (TWT-203).
As CFI-402257 progresses through clinical trials, correlative analyses will permit the characterisation of biomarkers and guide patient stratification.Our group has identified a two-gene expression signature within the APC components (ANAPC4 and CDC20) that is strongly correlated with CFI-402257 activity in TNBC cell lines. 184oreover, we have observed an increased cytotoxicity in CDK4/6i-resistant models harbouring RB1 loss. 155,184,193In light of this body of preclinical and clinical knowledge, CFI-402257 was recently granted US FDA Fast Track Designation for further development both as monotherapy and in combination with fulvestrant for HR + /HER2 − -mBC patients who have progressed on CDK4/6i.Developed in parallel, Schöffski et al. 197 recently communicated results on the safety and preliminary antitumour activity of the first-in-human study of the intravenous TTKi S81694 in patients with solid tumours.Among 38 participants, the most common adverse events included fatigue (58%), anaemia (45%) and nausea (32%), with mild haematological toxicity including grade ≥3 neutropenia in 11% of patients. 197Among 35 efficacy-evaluable patients, ORR was 6% and DCR was 43%.RP2D, however, was not defined due to the sponsor's decision to stop enrolment to monotherapy and prioritise S81694 testing in combination with cytotoxic agents.

KIF18A
9][200] In BC, KIF18A expression levels are increased and associated with higher tumour grade, metastases and shorter survival. 201enetic ablation of KIF18A led to proliferation inhibition via centrosome fragmentation and mitotic arrest both in vitro and in vivo, primarily affecting tumour cells with chromosome instability while inducing relatively low toxicity in diploid cells. 198,199,201These findings, combined with promising preclinical results in other tumour types, have paved the way for the development of pharmacological inhibitors such as sovilnesib and VLS-1488, currently undergoing phase 1 testing with no clinical data yet reported. 200,202 I G U R E 3 Rationale and challenges for the potential development of cell cycle inhibitors in combination with other therapies in breast cancer.ADC, antibody-drug conjugate; HRD, homologous recombination deficiency; ICI, immune checkpoint inhibitor; ILD, interstitial lung disease; LFT, liver function tests; TME, tumour microenvironment.

PERSPECTIVE OF CELL CYCLE INHIBITORS IN THE CURRENT THERAPEUTIC LANDSCAPE
Given the rapid introduction of novel endocrine agents and antibody-drug conjugates (ADCs) into the therapeutic landscape, the successful development of novel agents targeting the cell cycle will likely require robust anti-tumour activity, manageable toxicity and, in contrast to the initial use of CDK4/6i in combination with ET, biomarker enrichment.We envision four areas of strategic interest (Figure 3).

6.1
Targeting of cell cycle components either concurrently or upon resistance to CDK4/6i in HR + /HER2 − -mBC Important advances in the treatment of HR + /HER2 − -mBC have recently focused on targeting ESR1 mutations, where oral SERDs have demonstrated activity and ctDNAguided switching of the endocrine partner contributes to disease control in the face of emerging AI resistance. 203,2046][207] Most genomic alterations described in CDK4/6i-resistant tumours converge on their ability to override the G1/S checkpoint.Blocking CDK2 or CDK7, known to underlie CDK4/6i resistance, have attracted attention as potential strategies to restore sensitivity.In this regard, switching CDK4/6i or switching to other CDKi such as the CDK2/4/6i ebvaciclib and CDK7i samuraciclib could have a role, and have shown some activity in the post-CDK4/6i setting; however, much remains to be learned about the predictors of efficacy for these strategies, the optimal agents and endocrine combinations, and how they might compare.While samuraciclib is currently being tested in combination with newer endocrine agents in the post-CDK4/6i setting, efforts to elucidate the benefit of targeting CDK2 and CDK4 separately or together post-CDK4/6i, CDK2i combined with CDK4/6i, or CDK4i in treatment-naive patients are underway (Table 1).These trials will provide valuable data not only to understand CDK2, CDK4 and CDK7 targeting but also the optimal treatment sequencing or correlatives of benefit.
Encouraging preclinical evidence and manageable toxicity profiles in phase 1 trials support the development of PLK4i, AURKAi and TTKi in CDK4/6i-resistant HR + /HER2 --mBC.Notably, TTKi CFI-402257 has shown enhanced preclinical activity in models harbouring RB1 loss, a key mechanism of CDK4/6i resistance, and both TTKi and AURKAi seem to be tolerable in combination with fulvestrant and paclitaxel.Due to overlapping myelosuppression, the development of combinations of these compounds with CDK4/6i in the front-line setting is unlikely; however, an understanding of the clinical activity in the face of emerging resistance could support innovative approaches to develop sequential strategies.In the light of upfront ADC use post-CDK4/6i, PLK4i, AURKAi or TTKi are most likely to thrive in combination with SERDs and novel ETs as chemo-sparing regimens in molecularly selected populations, or in monotherapy as a later line of therapy.

6.2
Inducing synthetic lethality in combination with DDR agents such as PARPi or ATRi in TNBC or HRD disease PARPi olaparib and talazoparib are the only DDR agents approved for HER2 − -mBC patients with gBRCA1/2 mutations.Many others are currently in development with the goals of overcoming PARPi resistance, expanding efficacy against tumours with somatic DDR mutations and HRD and reducing toxicity. 208,209The interplay of DDR and cell cycle vulnerabilities provides an appealing therapeutic opportunity.Most TNBC harbour DDR and p53 defects that result in an inactive G1/S checkpoint, thus increasing the relevance of the G2/M checkpoint to respond to DNA damage. 1261][212][213][214][215] Increasing replication stress and genomic instability by interfering with cell cycle checkpoints may thus be leveraged to generate synthetic lethality or to re-sensitise tumours to DDR-based approaches.
7][218] This approach is currently being explored in the HOPE study, which combines olaparib, palbociclib and fulvestrant in BRCA1/2-mutant HR + /HER2 − -mBC patients.Despite the preclinical and clinical rationale, a substantial burden of haematological toxicity is likely.In small studies, response rates of 10−25% have been reported with WEE1i adavosertib or unselective CDKi selicilib in patients carrying gBRCA1/2 mutations. 65,128However, combinatorial strategies have been challenged by considerable haematological toxicity, with some examples including the combination of adavosertib with PARPi olaparib, or the combination of adavosertib, PLK1i volasertib and unselective CDKi with platins. 62,132,172,219pportunities may emerge with more tolerable DDR agents such as PARP1i and ATRi.The synthetic lethality of PARPi largely relies on PARP1 inhibition, with PARP2 being essentially involved in haematological homeostasis.Thus, novel PARP1-selective inhibitors such as AZD5305 or AZD9574 that are associated with less myelotoxocity may be more favourable partners for combination testing with cell cycle inhibitors.0][221][222] Since WEE1 and PKMYT1 are direct downstream transducers of ATR-mediated DDR, inhibitors of these molecules are well positioned for a cooperative effect.In this regard, the combination of adavosertib with ceralasertib or with an inhibitor of the anti-apoptotic protein BCL-XL led to synergistic antitumour effects with a significant therapeutic window in vivo and in a TNBC cell model, respectively. 223,224Furthermore, the genetic abrogation of PKMYT1 has been shown to restore sensitivity to adavosertib in vitro, and a phase 1 clinical trial investigating the combination of the PKMYT1i RP-6306 with ATRi RP-3500 is underway. 225

Combination of ADCs and cell cycle inhibitors
While additive or synergistic effects are plausible, preclinical evidence testing this strategy is lacking that could shed light into cell cycle synergies or enhanced genomic instability.The improved therapeutic index of ADCs over standard chemotherapies and their activity on selective tumour populations make them suitable partners for targeted agents, thus opening several therapeutic opportunities.First, the combination of cell cycle inhibitors with chemotherapies is feasible as demonstrated by the tolerability of the combination of paclitaxel with TTKi CFI-402257 and BAY1217389, and with AURKAi alisertib. 160,191,195One can expect a higher efficacy and less toxicity from the combination of TTKi or AURKAi and microtubule-targeting ADCs in development.Second, ADCs carrying DNA-damaging agents that act on the Sphase and lead to G2 arrest (e.g.topoisomerase inhibitors, anti-metabolites such as gemcitabine) could be suitably coupled with targeted inhibition of G2/M, as a way to render cytotoxicity by producing catastrophic genomic aberrations.Third, the sequential use of ADCs and cell cycle inhibitors, where ADCs may eradicate resistant clones and cell cycle inhibitors provide maintenance therapy thereafter.Modern ADCs with stable linkers, such as trastuzumab deruxtecan and datopotamab deruxtecan that cause substantially less myelosuppression than their counterparts may well provide exploratory candidates, although we expect this list to grow with the progressive optimisation of ADC properties and molecular selection of target populations.Finally, for the potent cell cycle inhibitors that have limited therapeutic index, the possibility of formulating these as ADCs may enable the development of molecularly-guided payloads that exploit cell cycle anomalies and overcome resistance to known agents.

Leveraging an increased tumour immunogenicity in combination with immunotherapeutics
7][228] The modest efficacy of anti-PD-1 therapy in the presumably "cold" HR + /HER2 − breast tumours, the niche of CDK4/6i, provides an opportunity to investigate the immune-priming effects of cell cycle inhibition as well as the features associated with acquired resistance that could enhance sensitivity to such strategies.In the study conducted by Yuan et al., 229 23 HR + /HER2 − -mBC patients received palbociclib, pembrolizumab and letrozole, achieving an ORR of 55% that included five complete responses out of 16 patients treated as firstline.Moreover, intriguing results were observed in the PACE study, a randomised phase II study where the switch of AI to fulvestrant after progression to AI and CDK4/6i did not improve PFS, but where addition of avelumab to fulvestrant and CDK4/6i nearly doubled ORR and PFS. 49The combination of abemaciclib plus pembrolizumab has also demonstrated anti-tumour activity, but caused high rates of interstitial lung disease and severe transaminase elevations which precluded further development, and similarly the phase Ib trial studying the combination of ribociclib and anti-PD-1 spartalizumab has been terminated due to undisclosed safety implications. 230,231reclinical evidence underscores the interest of blocking other cell cycle components to sensitise tumours to immune-checkpoint inhibition.PLK1i volasertib and WEE1i adavosertib have been shown to enhance response to anti-PD-1 agents in models of lung cancer by increasing PD-L1 expression, activating interferon pathways and stimulating cytotoxic T cell infiltration, and our group observed a similar outcome with TTKi CFI-402257 in a murine model of colon cancer. 192,232,233Furthermore, CFI-402257 was recently found to restore anti-PD-1 efficacy in a model of KRAS-LKB1-mutant lung cancer, which are intrinsically resistant to PD-1 blockade via epigenetic abrogation of STING. 234There are, however, limited clinical data testing this strategy, with the combination of the unselective CDKi dinaciclib and pembrolizumab, and of CFI-400945 and durvalumab, being safe but exhibiting limited activity in unselected patients with heavily pre-treated mTNBC. 69,111

CONCLUSION
The deregulation of the cell cycle enables BC cells to proliferate and thrive despite adverse environments and cumulative genomic instability.Both ER-dependent and -independent mechanisms modulate cell cycle checkpoints, where CDK4/6 inhibition has attained remarkable success in HR + tumours.Despite its appeal as a pharmacological target, the complexity of the cell cycle, with pleiotropic regulatory pathways and functional redundancy of its components, has hindered therapeutic development.Our growing understanding of the science underlying cell cycle deregulation along with advances in drug design provide an expanded therapeutic window for clinical development.The successful incorporation of CDK4/6i in metastatic and now early HR + /HER2 − -BC has created the new entity of CDK4/6iresistant disease, with unique treatment-resistant genomic alterations.Bolstered by strong preclinical evidence and careful consideration of molecular determinants of sensitivity, novel inhibitors may be well-positioned to overcome CDK4/6i resistance, induce synthetic lethality in genomically unstable tumours and boost immunogenicity and sensitivity to immunotherapeutics, either as monotherapy or combined with CDK4/6i, DDR agents or immune checkpoint inhibitors, respectively.However, the therapeutic landscape is rapidly expanding with the introduction of novel endocrine agents and ADCs post-CDK4/6i for HR + /HER2 − disease, and of immune-chemotherapy combinations and ADCs in TNBC.Demonstrating the clinical benefit of these novel cell cycle inhibitors will require rational, science-driven clinical trial designs and comprehensive efforts to identify predictive biomarkers.